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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 368-371
A Clinical Trial of Retroviral-Mediated Transfer of a
rev-Responsive Element Decoy Gene Into CD34+
Cells From the Bone Marrow of Human Immunodeficiency Virus-1-Infected
Children
By
Donald B. Kohn,
Gerhard Bauer,
C. Robert Rice,
J.C. Rothschild,
Denise A. Carbonaro,
Penelope Valdez,
Qian-lin Hao,
Chen Zhou,
Ingrid Bahner,
Karen Kearns,
Kate Brody,
Sarah Fox,
Elizabeth Haden,
Kathy Wilson,
Cathy Salata,
Cathy Dolan,
Charles Wetter,
Estuardo Aguilar-Cordova, and
Joseph Church
From the Division of Research Immunology/Bone Marrow Transplantation
and the Division of Immunology/Allergy, Childrens Hospital Los Angeles,
the Department of Pediatrics, University of Southern California School
of Medicine; and Gene Vector Laboratories, Texas Children's Cancer
Center, Baylor College of Medicine, Houston, TX.
 |
ABSTRACT |
Genetic modification of hematopoietic stem cells with genes that
inhibit replication of human immunodeficiency virus-1 (HIV-1) could
lead to development of T lymphocytes and monocytic cells resistant to
HIV-1 infection after transplantation. We performed a clinical trial to
evaluate the safety and feasibility of this procedure, using bone
marrow from four HIV-1-infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34+ cells,
performed in vitro transduction with a retroviral vector carrying a
rev-responsive element (RRE) decoy gene, and reinfused the
cells into these subjects with no evidence of adverse effects. The
levels of gene-containing leukocytes in peripheral blood samples in the
1 year after gene transfer/cell infusion have been extremely low. These
observations support the potential of performing gene therapy for HIV-1
using hematopoietic cells, but emphasize the need for improved gene
transfer techniques.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
A POTENTIAL THERAPEUTIC approach to human
immunodeficiency virus-1 (HIV-1) infection is the genetic modification
of cells of a patient to make them resistant to HIV-1, termed
intracellular immunization.1,2 Gene therapy for HIV-1 could
target either mature peripheral blood T lymphocytes or hematopoietic
stem cells. While T lymphocytes are more readily isolated and
transduced, hematopoietic stem cells may be an attractive target
because of their theoretical ability to generate a broad repertoire of
mature T lymphocytes, as well as the monocytic cells (macrophages,
dendritic cells, and microglia), which are also involved in HIV-1
pathogenesis. We have previously shown that overexpression of HIV-1
rev-responsive element (RRE) sequences, as part of the
transcript from the long-terminal repeat (LTR) of a retroviral vector,
led to inhibition of HIV-1 replication in human T lymphocytes and in
monocytic cells derived from transduced CD34+ hematopoietic
progenitor cells. There was no evidence that expression of the RRE
decoy adversely affected cell function.3,4
| |
STUDY DESIGN |
We have performed a pilot clinical trial to assess the safety,
feasibility, and efficacy of retroviral-mediated transfer of the RRE
decoy gene into CD34+ cells isolated from the bone marrow
of HIV-1-infected pediatric subjects. The protocol and informed
consent document were reviewed by the Committee on Clinical
Investigations (CCI) and the Institutional Biosafety Committee at
Childrens Hospital Los Angeles, the Recombinant DNA Advisory Committee
of the National Institutes of Health, and the Center for Biologics
Evaluation and Review of the Food and Drug Administration.
Subjects were recruited from the Pediatric AIDS Program at Childrens
Hospital Los Angeles. No specific criteria for CD4+ cell
count or HIV-1 viral loads were used, to allow enrollment of subjects
with a range of disease severity. Informed consent was obtained from
parents and subjects 12 years or older or assent from subjects 7 to 11 years of age, following the guidelines of the CCI. Screening studies
were performed to exclude subjects with severe organ dysfunction or
contraindications to general anesthesia for bone marrow harvest. Bone
marrow aspirates (5 to 10 mL) were obtained to determine the frequency
of CD34+ cells and to grow autologous bone marrow stromal
cells for support of the CD34+ cells during transduction.
All subjects met the inclusion criteria of  1% CD34+
cells in the marrow aspirate. The anti-retroviral drug regimens that
the subjects received were those previously prescribed by their primary
physicians and were not influenced by participation in the study.
| |
RESULTS AND DISCUSSION |
Four HIV-1+ pediatric subjects were enrolled and underwent
the gene transfer protocol. Their age, weight, absolute
CD4+ T-lymphocyte count, and plasma HIV-1 viral load are
shown in Table 1. Subjects no. 1, 2, and 4 were teenagers who had been HIV-1+ for 12 to 16 years,
after infection via transfusion. Subject no. 3 was an 8-year-old who
was infected perinatally. Two of the subjects had very low
CD4+ cell counts (2 and 6 CD4/µL) and two had
CD4+ cell counts between 250 and 450/µL. All
had high viral loads, despite two or three anti-retroviral drug
therapy.
Bone marrow was obtained under general anesthesia from the bilateral
posterior iliac crests and collected into RPMI 1640 with 500 U/mL
heparin. Subjects remained in hospital overnight on the General
Clinical Research Center after marrow donation and were discharged to
home the next day without complications. The bone marrow was processed
by centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech AB,
Uppsala, Sweden), followed by CD34+ cell isolation using
the Isolex 300i (Nexell Therapeutics Inc, Irvine, CA).
Table 1 shows the volumes of bone marrow collected and cell yields. The
target volume of 10 mL/kg was reached in subjects no. 1 through 3, but not in subject no. 4. The unprocessed marrow samples contained
between 1.4% and 3.0% CD34+ cells, reconfirming in each
case that the subjects met the inclusion criteria for marrow
CD34+ cell content of 1%. The cell products produced by
the Isolex 300i contained between 69.7% and 83.7% CD34+
cells. The two subjects with extremely low CD4+ cell counts
(nos. 2 and 4) had lower cell yields than the two subjects with higher
CD4+ cell counts, although the small number of subjects
preclude any firm conclusions.
To determine whether T lymphocytes expressing the RRE decoy have
improved survival, we used an internal comparative marking technique,
as has been used by others.5,6 One half of each patient's
cells were transduced with the L-RRE-neo vector that contains a 41-bp
portion of the HIV-1 RRE sequences, inserted immediately upstream of
the neo gene.3 The other half of their cells were
transduced with the control LN vector. Both vectors were packaged as
clones in PA317 amphotropic packaging cells.7 Clinical
grade supernatants were produced by the Gene Vector Laboratory at
Baylor College of Medicine in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. Titers of the
supernatants were 1 to 3 × 106/mL based on
G418-resistance of serial dilutions on 3T3 cells. Testing for
adventitious agents and replication-competent retroviruses (RCR) were
performed by the Gene Vector Laboratory at Baylor College of Medicine
or by Microbiologic Associates (Rockville, MD) and were all negative.
The CD34+ cells were divided into two portions of equal
cell numbers, one for transduction with L-RRE-neo and one for
transduction with LN. Autologous bone marrow stromal cells (irradiated
with 2,000 cGy, 1 day before use) were used as support layers during transduction of cells from subjects no. 1 through 3.8 For
subject no. 4, recombinant fibronectin CH-296 (RetroNectin; Takara
Shuzo Co, Ltd, Otsu, Shiga, Japan) was used instead of autologous
stroma. CD34+ cells were transduced for three 24-hour
transduction cycles with recombinant cytokines interleukin-3 (IL-3) (20 ng/mL; Novartis, Piscataway, NJ), IL-6 (50 ng/mL; Novartis), and Steel
factor (100 ng/mL; R & D Associates, Minneapolis, MN) and
protamine sulfate (4 µg/mL; Lyphomed, Deerfield, II), as
described previously.4 Because subject no. 4 had a poor
yield of marrow and CD34+ cells, no conclusions can be
drawn about any differences in cell recovery or transduction
using stroma versus fibronectin CH-296.
After the three transduction cycles, the nonadherent cells and culture
medium were collected from the flasks, and then the adherent cells were
collected from the flasks using enzyme-free cell dissociation buffer
(GIBCO-BRL, Bethesda, MD). The nonadherent and adherent cells were
combined and washed four times with Hanks' balanced salt solution with
1 U/mL heparin and resuspended in 15 mL plasma-lyte A with 1 U/mL
heparin. The two cells pools transduced separately with L-RRE-neo and
LN were combined for a total final volume of 30 mL, which constituted
the final cell product. Patients were monitored clinically during the
cell infusion and afterward overnight.
All monitoring gram stains, bacterial, fungal, and mycoplasma cultures,
endotoxin assays, and measurements for RCR in culture medium and cell
pellets were negative. There was no detectable HIV-1 in the transduced
CD34+ cell pellets or culture medium by DNA polymerase
chain reaction (PCR) or reverse transcriptase (RT)-PCR, respectively.
Subjects received their cells by intravenous infusion without
complications, based on clinical monitoring and laboratory studies. Postinfusion evaluations showed no perturbations in vital signs, complete blood count (CBC), or chemistry panels. There was
no evidence that the patients were exposed to RCR, by assay for
infectious RCR from their peripheral blood mononuclear cells (PBMC)
(days +1, +7, and +30) or by Western blot analysis for the development of serum antibodies reacting against Moloney murine leukemia virus (MoMuLV) proteins (months +1, +2, +3). There were no
detectable changes in any of the subject's plasma HIV-1 levels from
the marrow harvest or cell reinfusion. Thus, the subjects did not
receive a significant inoculum of HIV-1 upon reinfusion of their
transduced cells. Apparently, the transduced cells do not serve as a
site of HIV-1 replication in vivo, despite their activation in vitro from culture with the recombinant cytokines used to stimulate stem cell
proliferation to allow retroviral-mediated transduction.9
Transduction of clonogenic progenitor cells within the
CD34+ cells was evaluated by growing CFU-C colonies and
measuring the percentage that were G418-resistant and the percentage
that contained vector DNA by DNA-PCR of individual colonies which were
not selected in G418 (Table 1). Transduction efficiencies were between
7% and 30% by either assay and the percentages of transduced
colony-forming unit-cells (CFU-C) measured by either G418-resistance or
PCR were not significantly different (P = .77).
The two vectors (L-RRE-neo and LN) led to similar extents of
transduction (data not shown, P = .87).
Venous blood samples were obtained at serial times after the infusion
of transduced CD34+ cells to measure the percentage of PBMC
containing vector sequences. Positive samples have been seen in all
four subjects (Fig 1). Cells containing
both the L-RRE-neo and LN vectors were detected on the day after cell
infusion in two of the subjects, at frequencies of 0.03% and 0.01% (1 to 3 gene-containing cells per 10,000). With the exception of those day
+1 samples, all subsequent samples were negative for the L-RRE-neo
vector. Cells containing the LN vector were detected in some later
samples for as long as 270 to 330 days, at frequencies of 1 cell per
100,000.
View larger version (16K):
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| Fig 1.
Gene frequency in PBMC after infusion of transduced
CD34+ cells. Isolation of genomic DNA from patient
leukocytes and PCR amplification and analysis were performed as
described previously.11 To develop quantitative standards,
mixtures of genomic DNA extracted from CEM cells containing one
proviral copy of either the LN or the L-RRE-neo vectors were diluted in
genomic DNA extracted from nontransduced cells. These
standards plus multiple samples of DNA from nontransduced PBMC were
analyzed with each assay of patient samples. A single pair of PCR
primers was made to simultaneously detect the presence of sequences
from the LN and L-RRE-neo vectors. The 5' primer was complimentary to
the MoMuLV Psi region (5'-CGAGACCTCATCACCCAGGTTAAG-3' sense) and the 3'
primer was complimentary to the neo gene
(5'-CATCAGAGCAGCCGATTGTCTG-3' antisense). This pair of primers produces
a product of 391 bp from the LN vector (N) and 451 bp from the
L-RRE-neo vector (R); the increase in the L-RRE-neo product being
caused by the presence of the RRE sequences immediately 5' of the neo
gene, within the span of the primer pair. An oligonucleotide
(5'-TCGATCCTCCCTTTATCCAGCC-3') complimentary to a sequence present in
the resultant PCR products from each vector was labeled with
 -32P-dATP and used as probe.
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The failure to achieve significant transduction and engraftment of
hematopoietic stem cells in this trial stands in contrast to our
results using similar methods of gene transfer into CD34+
cells from the umbilical cord blood of infants with adenosine deaminase
(ADA)-deficient severe combined immunodeficiency
(SCID).10,11 In the ADA-deficient subjects, the continued
presence of gene-containing cells of myeloid and lymphoid lineages for
over 5 years showed that long-lived stem cells were transduced and
engrafted. The difference may reflect the presence of a modest
percentage of hematopoietic stem cells in cord blood which are actively
undergoing cell replication, in contrast to the near-absence of
replicating stem cells in bone marrow.12 The spontaneously
dividing stem cells in umbilical cord blood would be susceptible to
transduction by MoMuLV retroviral vector, whereas the quiescent stem
cells from bone marrow would not. It is also possible that immunologic responses to either the neomycin phosphotransferase protein encoded by
the vectors or to components of the cell culture medium may have led to
in vivo elimination of the transduced cells.
Recent incremental improvements in retroviral-mediated gene transfer
into human hematopoietic stem cells (HSC) have been achieved, using
gibbon ape leukemia virus (GALV) pseudotypes, "mobilized bone
marrow," recombinant fibronectin support, new cytokines (Flt-3 ligand, thrombopoietin), and manipulation of cell-cycle
kinetics.13-18 Combinations of these
techniques have resulted in modest, yet significant, increases in gene
marking in primate stem cell transplant models (eg, 10%, up from the
previous ceiling of 0.1% to 1.0%).19,20 However, even
higher levels of gene transduction of stem cells are likely to be
needed for applications to many genetic diseases and acquired
immunodeficiency syndrome. The development of vectors based on
lentiviruses, such as HIV-1 or feline immunodeficiency virus (FIV),
holds the promise of producing increased transduction of HSC, due to
their ability to transduce quiescent cells such as neurons,
hepatocytes, and macrophages.21,22 The safety
and feasibility shown by the present study will allow future studies to
evaluate these newer methods for their potential efficacy.
| |
ACKNOWLEDGMENT |
We thank the patients and families who participated in this study.
Essential reagents were generously provided by Katie Harding (Novartis,
Piscataway, NJ), John MacManus and Virginia Mansour (Baxter/Nexell,
Irvine, CA), and Dr Ikunoshin Kato and Setsuko Yoshimura (Takara, Otsu,
Shiga, Japan).
| |
FOOTNOTES |
Submitted December 29, 1998; accepted March 30, 1999.
These studies were performed in the General Clinical Research Center at
Childrens Hospital Los Angeles (3 MO1 RR0043-35S1) with support by the
GCRC Gene Therapy Core Laboratory, a SPIRAT grant from the National
Institute of Allergy and Infectious Diseases (1U19 AI36606), and
research grants from the Pediatric AIDS Foundation (no. 30001-17-GT)
and the T.J. Martell Foundation. D.B.K. is the recipient of an
Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Donald B. Kohn, MD, Mailstop #62, Childrens
Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA 90027; e-mail:
dkohn{at}chla.usc.edu.
| |
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ABSTRACT
INTRODUCTION